Kinetochores are multi-protein complexes that assemble on centromeric (CEN) DNA and perform three essential functions in chromosome segregation (i) they bind paired sister chromatids to spindle microtubules (MTs) in a bipolar fashion compatible with disjunction at anaphase (ii) they move chromosomes back and forth along spindle MTs by coupling plus-end dynamics to chromosome movement during metaphase and anaphase (iii) they generate the spindle checkpoint signal that links anaphase onset to the prior completion of chromosome-MT attachment. The long-term goals of this study are to understand these three kinetochore functions in precise molecular terms. Biochemical, genetic and imaging experiments will be performed primarily in the budding yeast S. cerevisiae, an organism whose kinetochores are the simplest known, but key conclusions will also be confirmed in human tissue culture cells. : Aim 1. A key inner kinetochore complex comprising CEN DNA, sequence-specific DNA binding proteins, a specialized histone H3 (CenHS) and associated subunits will be reconstituted in vitro and subjected to detailed functional and biophysical analysis. Aim 2. The role of the inner kinetochore in organizing and assembling a "linker" layer comprising the NdcSO, COMA, MIND and Spc105 complexes will be studied in vitro and in vivo Aim 3. Kinetochore subassemblies active in MT attachment, and comprising DNA-binding and linker proteins in combination with one or more MAPs or motors, will be reconstituted in vitro for detailed analysis of force-generating activities. Aim 4. The role of human homologues of yeast linker proteins will be examined using RNAi-based protein depletion, live-cell imaging and biochemical fractionation. Aim 5. An on-line resource will be created to disseminate information on kinetochore functions, assays and phylogeny (a data sharing resource). The significance of these experiments derives from their aim of providing a functional and mechanistic understanding of a particularly simple kinetochore and of using this information to inform the analysis of kinetochores in human cells. Errors in kinetochore assembly cause chromosome instability (CIN) a nearly universal property of human tumor cells. The origins of CIN, and its precise role in tumor progression remain unknown, but functional studies represent one the best way to understand the origins of CIN and to determine whether its appearance in cancer cells is linked to tumorigenic potential. [unreadable] [unreadable] [unreadable]